Ralph M. Steinman

Introduction: Given their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC⁺ dendritic cells (DCs) in a mouse breast cancer model. Methods: We genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4⁺/CD8⁺ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated. Results: We demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4⁺ T cell immunity, CD8⁺ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4⁺ and CD8⁺ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice. Conclusions: Immunization of mice with HER2 protein vaccine targeting DEC⁺ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4⁺ and CD8⁺ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 μg of HER2 protein incorporated in the vaccine. Vaccine-induced CD4⁺ and CD8⁺ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.

Foxp3+ regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor β (TGF-β) and retinoic acid. We found that the CD11chigh dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3+ cells. The induced CD4+CD25+Foxp3+ cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RBhi T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3+ T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease.

This Viewpoint series provides authoritative and detailed outlines of exciting areas of DC research. Some of the subjects that frequently come up include development of DC; distribution of DC in lymphoid and non‐lymphoid tissues such as skin, intestine and lung; different forms or subsets of DC; and the role of DC in initiating tolerance and immunity. In this Preface, I will introduce the Viewpoints and consider some future challenges as well as the medical relevance of DC research.

DC present exogenous proteins to MHC class I‐restricted CD8⁺ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8⁺ T‐cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8⁺ or DEC‐205⁺ DC are specialized for cross‐presentation, and this subset can be increased 10‐fold in numbers following Fms‐like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross‐presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti‐CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11c^high DC, particularly the DEC‐205⁺ subset. DC cross‐present HIV gag to primed CD8⁺ T cells, but when the antigen is delivered within an antibody to DEC‐205 receptor, cross‐presentation becomes 100‐fold more efficient than non‐targeted antigen. This finding requires gag to be engineered into anti‐DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross‐presentation, since their use overcomes the obstacle posed by the low number of cross‐presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC‐targeted vaccines.

To develop a tumor vaccine directly targeting tumor antigen to dendritic cells in situ, we engineered human mesothelin (MSLN) into an antibody specific for mouse DEC‐205, a receptor for antigen presentation. We then characterized both T cell and humoral responses to human MSLN and compared immunizing efficacy of DEC‐205‐targeted MSLN to nontargeted protein after a single‐dose immunization. Targeting human MSLN to DEC‐205 receptor induced stronger CD4⁺ T‐cell responses compared to high doses of mesothelin protein. Approximately 0.5% CD4⁺ T cells were primed to produce IFN‐γ, tumor necrosis factor‐α, and IL‐2 via intracellular cytokine staining, and the T cells also could proliferate rapidly. The immune response exhibited breadth because the primed CD4⁺ T cells responded to at least three epitopes in the H‐2^b background. Targeting MSLN protein to DEC‐205 receptor also resulted in cross‐presentation to CD8⁺ T cells. Antibody responses against human MSLN were also detected in serum from primed mice by ELISA assays. In summary, targeting of MSLN to DEC‐205 improves the induction of CD4⁺ and CD8⁺ T‐cell immunity accompanied by an antibody response. DEC‐205‐targeting could be valuable for enhancing immunity to MSLN in cancers where this nonmutated protein is expressed.

This volume provides a small sampling of the rapidly growing science of cancer immunology. Growth in the field includes advances on the requirements for immunization or vaccination and the ways in which immunity can be suppressed or blocked, including active tumor‐based mechanisms. I would like to introduce the papers in this volume and then deal with a subject that pervaded many discussions among symposium participants. The subject is the need for a much better supported and organized effort to design optimal studies of immunology in cancer patients so that cancer vaccines can become a major means to prevent and treat this disease.

Dendritic cells (DCs) are professional APCs that can control immune responses against self and altered self, typically foreign, determinants. DCs can be divided into several subsets, including CD8α⁺ and CD8α⁻ DCs. These subsets possess specific functions. For example, mouse splenic CD8α⁺, but not CD8α⁻ DCs selectively take up dying cells and cross-present cell-associated Ags to naive T cells. In this study, we identified genes that were more expressed in CD8α⁺ than CD8α⁻ DCs by microarray analysis. Only one of these genes, when the extracellular domains were linked to human IgG Fc domain, could bind to late apoptotic or necrotic cells. This gene was a new member of the triggering receptor expressed on myeloid cells (Trem) family, Trem-like 4 (Treml4). Treml4 mRNA and protein, the latter detected with a new mAb, were predominantly expressed in spleen. Treml4, like other Trem family members, could associate with the adaptor molecule DNAX activation protein 12 kDa, but neither DNAX activation protein 10 kDa nor FcRγ. Consistent with the microarray data, we confirmed that Treml4 protein was more expressed on CD8α⁺ than CD8α⁻ DCs, and we also found that Treml4 was expressed at high levels on splenic macrophages in spleen, particularly red pulp and marginal metallophilic macrophages. In addition, Treml4 expression on DCs was not changed after maturation induced by TLR ligands. Thus, Treml4 is a new Trem family molecule that is abundantly expressed on CD8α⁺ DCs and subsets of splenic resident macrophages, and can recognize dead cells by different types of phagocytes in spleen.

Summary: The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC‐205/CD205 marker (‘DEC’), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti‐DEC and anti‐CD3 showed the expected concentration of DEC⁺ cells in the relatively small T‐cell areas of the human spleen. DEC⁺ cells were also found in other regions of the white pulp. In all regions, the DEC⁺ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7‐2/CD86 costimulator and DC‐lysosome‐associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC‐specific ICAM‐3 grabbing non‐integrin (SIGN)/CD209, and just inside these cells were DEC⁺ profiles. The DEC⁺ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti‐DEC‐205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.